1. The expression of α-tubulin、β-tubulin、n-luciferase、c-luciferase

1.1 vector construction

We got gene fragments of α-tubulin、β-tubulin、n-luciferase、c-luciferase via PCR and verified them by electrophoresis(fig.) The theoretic gene size of α-tubulin is 1356bp, β-tubulin is 1335bp, n-luciferase is 1248bp, c-luciferase is 459bp, which fit our experimental results.

We ligated our gene fragments to E.coli expression plasmid pET30a(+) and the sequencing result showed that we successfully constructed the α-tubulin, β-tubulin, n-luciferase, c-luciferase expression vectors.

We transformed the expression vectors to E.coli expression strain TranB(DE3). After culturing and inducing with IPTG, we lysed the bacteria and used SDS-PAGE/ western-blot to test the protein from the bacteria supernatant, pellet and the renatured inclusion body.

Apart from this, we also transformed plasmids to Rosetta(DE3) which can express rare codons and improve the expression level of eukaryotic protein.

Based on the results, α-tubulin and β-tubulin were successfully expressed in cell.

We collaborated with Fujian Agriculture and Forest University and asked them to test the interaction between α and β-tubulin. Thus verified the activity of tubulin monomers.

这里放发福的结果

2. The expression of fusion protein

2.1 PCR fusion PCR

We constructed α-tubulin-YNE、YNE-α-tubulin、α-tubulin-YCE、YCE-α-tubulin、β-tubulin-YNE、YNE-β-tubulin、β-tubulin-YCE、YCE-β-tubulin、α-tubulin-nluc、nluc-α-tubulin、α-tubulin-cluc、cluc-α-tubulin respectively via fusion PCR. After ligating these fusion gene fragments to TransB(DE3), we transformed target plasmids to Trans5α. When colony PCR was done, we picked correct colony for plasmid amplification.

我们将PCR验证结果正确的载体送去华大公司测序，测序结果表明我们成功构建出α-tubulin-YNE、YNE-α-tubulin、α-tubulin-YCE、YCE-α-tubulin、β-tubulin-YCE、YCE-β-tubulin、α-tubulin-nluc、cluc-α-tubulin表达载体。

We transformed the expression plasmids to E.coli expression strain TranB(DE3). The protein expression predicted website http://www.biotech.ou.edu/ showed that our fusion protein would probably expressed as inclusion bodies. We therefore renatured the inclusion bodies and verified through SDS-PAGE.

2.2 Gateway

Apart from these, we also expressed our target protein through Rosetta(DE3).

In our experiment, we also try to construct fusion protein vectors with Gateway Large-scale Cloning technology. We used Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector. Designing primer based on β-tubulin sequence and running PCR procedure. From this picture, the band of β-tubulin was correct.

In order to do LR reaction, we used the restriction endonuclease Not I to digest the entry vector. The bands of linear vector and the origin vector suggested that the digestion was efficiency.

Using Invitrogen Gateway LR Clonase II Enzyme Mix, the entry vector can be ligate with pCambia1300-nluc and pCambia1300-cluc respectively. So the destination vectors were complete. After transformation, running PCR with β-tubulin’s primers, the bands show high positive rates as showed in figure 2.2.3.

Extracting plasmid, the product gal bands show that cluc-β-tubulin is correct. Running PCR using the reverse primer of β-tubulin and the forward primer of cluc, the correct band existed.

3. 体外提取微管蛋白结果